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A species-specific polymerase chain reaction (PCR) was developed for the identification of beef that is cattle (Bos indicus) meat by targeting mitochondrial D-loop region. A pair of beef-specific primers was designed to amplify conserved region of D-loop and possibility of cross-amplification was ruled by taking into account other species comprising of mammals, birds, rodents and fish. The amplified PCR product of 381 bp was evident in raw (n = 20), cooked (60, 80 and 100°C), autoclaved (121°C) and micro-oven processed beef samples. Sensitivity of PCR assay was at 0.1% for detection of beef adulteration and the limit of detection (LOD) of beef DNA was 0.1 picograms (pg). Newly developed beef-specific PCR assay was rapid and sensitive and hence could be applied for the detection of adulteration of meat. [ABSTRACT FROM AUTHOR]